Non-t cell binding peptides and their uses

ABSTRACT

The present invention provides a non-T cell binding peptide and its analogs used for the treatment of rheumatoid arthritis. The polypeptide therapeutic agent can specifically inhibit abnormal immune responses of the rheumatoid arthritis, and fundamentally control the progression of this disease with effect on the initiating factor of the disease development.

FIELD OF THE INVENTION

The present invention relates to the preparation of non-T cell bindingpeptides and their uses as drugs for the treatment of rheumatoidarthritis and other autoimmune diseases.

BACKGROUND

Rheumatoid arthritis (RA) is a kind of chronic autoimmune disease mainlycharacterized by joint destruction and deformity, which can disablepatients. With the morbidity of 0.34% in China, there are nearly fivemillion patients in the whole country, and the percentage of disabilityreaches up to 93%. The procedure of the onset of the disease is an“initiation—linked immune response” procedure driven by antigens. Immunedamage(s) mediated by infection factor(s) and autoimmune response(s) arethe basis for the onset and progression of rheumatoid arthritis. Antigenpolypeptides are expressed through antigen presenting cells in vivo, andthey activate T lymphocyte, which results in the release of cytokinesand the increase of production of inflammatory factors, such as immuneglobulins, chemokines and free radicals, which consequently inducetypical pathologic changes of rheumatoid arthritis, includingvasculitis, synovium hyperplasia, damage(s) of cartilages and bones.

Currently, there is still no drug which can completely controlrheumatoid arthritis. Generally, non-steroidal anti-inflammatory drugssuch as Brufen and declofenic acid are used clinically for temporarilyrelieving symptoms. Disease-modifying anti-rheumatoid drugs, such asmethotrexate and leflunomide, are used to extensively inhibit immuneresponses by suppressing DNA synthesis and thereby inhibit jointinflammation. Therefore, such drugs do not take effect on the initialprocedure of the disease onset in the treatment of rheumatoid arthritis,and it is very difficult for them to control the disease completely,which results in the continuous progress of the systemic and jointdamages, and disability at last. Moreover, because of the extensiveimmune suppression of these drugs, there are a lot of side effects, suchas myelosuppression and abnormal hepatic functions, and therefore thesedrugs cannot be administered to many patients.

At present, there is an urgent need for drugs which can take effect onthe etiology of rheumatoid arthritis and treat this disease bysuppressing the initial procedure of immune responses. Thus after manyyears of research, the inventors found the non-T cell binding peptides,which can be used for treating rheumatoid arthritis. It proves in ourresearch that the peptides of the present invention can inhibit therecognition to antigens of HLA-DRβ1 molecules and T cells, and therebyinhibit the consequent autoimmune responses. As such, they can preventand control the onset and development of the disease through the keyprocedure of the onset of rheumatoid arthritis.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a non-T cell bindingpeptide, which can be used to treat rheumatoid arthritis effectively.

Another object of the present invention is to provide the use of saidnon-T cell binding peptide in the treatment of rheumatoid arthritis.

Finally, the present invention provides a pharmaceutical composition(s)comprising the said non-T cell binding peptide.

The term “non-T cell binding peptide” used herein refers to the peptideswhich are produced by altering one or more amino acids of SEQ ID NOs:1-7 and which can still bind to HLA-DRβ1 after alteration. The methodsfor altering amino acid sequences are well known in the art.

Generally, the present invention includes: one or more amino acidsequences which can bind to T cell receptor and which can stimulate Tcell proliferation in wild type CII peptide are substituted with Alanine(A) or Glycine (G), and the amino acids which can bind to HLA-DRβ1 areremained, thus producing a group of novel CII polypeptide moleculeswhich can only bind to HLA-DRβ1 molecules and cannot be recognized by Tcell receptors, i.e., the seven non-T cell binding peptides listed inthe attachment (267A, 268A, 269A, 270A, Mut 269-270, Mut 268-270, Mut267-270). Their specific sequences and IDs are shown in the SequenceListing section.

The recognition and binding to each other among the three molecules.HLA-DRβ1, antigen peptide and T cell receptor, is the key procedure ofabnormal immune responses in rheumatoid arthritis. Therefore, the objectof the present invention is to block the binding of T cell(s) toantigen(s), which is the key point of the etiology of rheumatoidarthritis, and suppress the in vivo abnormal immune responses inpatients with rheumatoid arthritis by utilizing the recognition of Tcells to antigens and the competitive inhibition of non-T cell bindingpeptides to pathogenic antigens, and thereby control the progress ofonset and development of the disease so as to reach the goal of treatingrheumatoid arthritis completely.

It proves that amino acids 70-74 in HLA-DRβ1 contain a consensussequence of QK/RRAA (i.e. Gln-Lys/Arg-Arg-Ala-Ala,glutamine-lysine/arginine-arginine-alanine-alanine)⁽¹⁾. This consensussequence is related to the formation of HLA-DRβ1-antigen binding cleftand is the functional amino acids which function in the binding toantigens. Plenty of research by the inventors and Wucherfennig et alproved that positively charged amino acid 71 (Lys or Arg) in thissequence is the key site of antigen binding⁽²⁻⁸⁾.

By research on crystal structure of HLA-DRβ1-antigen dimmer using X-raydiffraction technique, it is found that a variety of antigen peptideswhich bind to rheumatoid arthritis related HLA-DRβ1 (DR4/DR1) moleculesare extremely similar in configuration, including denatured type IIcollagen (CII) and Heat Shock Protein (HSP) etcetera⁽⁹⁻¹²⁾. From thetridimensional structures of these peptides (FIG. 1), it can be seenthat the side chains of Phe₂₆₃ (P 1), Glu₂₆₆ (P4) and Gly₂₇₁ (P9)stretch to the HLA-DRβ1 molecule in the left, and are imbedded into theantigen binding cleft entirely or partially, while side chains of theother amino acids stretch to another side or the side opposite toHLA-DRβ1 (the side of T cell receptor) to stimulate T cell activation.From FIG. 2, it can be seen that the side chains of P1, P4, P9 of CIIpolypeptide are imbedded into the antigen binding “pocket” of HLA-DRβ1.Negatively charged P4 (Glu) is adjacent to positively charged amino acid71 (Lys₇₁) of HLA-DRβ1, which forms the polar binding with highaffinity. Therefore, Glu₂₆₆ might be the key amino acid of CIIpolypeptide which binds to DRβ1⁽¹³⁻¹⁴⁾. In further research, it provesthat CII bind to T cell receptors mainly through Phe₂₆₃, Gly₂₆₅, Glu₂₆₆and Gly₂₇₁, Glu₂₇₂, and thus activates T cells. From this we can seethat the major HLA-DRβ1 binding amino acids in CII polypeptide arePhe₂₆₃, Gly₂₆₅, Glu₂₆₆ and Gly₂₇₁, Glu₂₇₂, whilst the major T cellreceptor (TCR) binding amino acids are Gln₂₆₇, Gly₂₆₈, Pro₂₆₉ and Lys₂₇₀(Table 1). TABLE 1 Amino acids binding to T cell receptors and HLA-DRβ1in CII DRβ1     DRβ1  DRβ1                 DRβ1 DRβ1  F   K   G   E   Q  G   P   K   G   E 263 264 265 266 267 268 269 270 271 272                TCR   TCR   TCR   TCR* F = Phe (Phenylalanine), K = Lys (Lysine), G = Gly (Glycine), E = Glu(Glutamate), Q = Gln (Glutamine), P = Pro (Proline), A = Arg (Arginine),A = Ala (Alanine).

Based on the aforementioned research, the inventors produced a non-Tcell binding peptide which can only bind to DRβ1 molecules and whichcannot be recognized by T cell receptors by substituting the amino acidswhich can bind to T cell receptors and which can stimulate T cellproliferation in CII peptide with Alanine (A) and Glycine (G), andremaining the amino acids which can bind to HLA-DRβ1. The non-T cellbinding peptides can specifically bind to HLA-DRβ1, and competitivelyinhibit the binding of autoantigens to HLA-DRβ1, and thus inhibitHLA-DRβ1 mediated autoimmune responses and inhibit so induced pathologicprocesses, such as release of inflammatory factors.

Therefore, in the embodiments of the present invention, non-T cellbinding peptides and their analogs are provided. The following peptidesare preferred: F K G E A G P K G E (SEQ ID NO: 1), F K G E Q A P K G E(SEQ ID NO: 2), F K G E Q G A K G E (SEQ ID NO: 3), F K G E Q G P A G E(SEQ ID NO: 4), F K G E Q G A A G E (SEQ ID NO: 5), F K G E Q A G A G E(SEQ ID NO: 6) and F K G E G A G A G E (SEQ ID NO: 7). Thesepolypeptides can bind to the specific sequence of QK/RRAA (i.e.Gln-Lys/Arg-Arg-Ala-Ala) in HLA-DRβ1 which is related to the onset ofrheumatoid arthritis, and can thereby inhibit T cell activation, andconsequently reach the goal of treating rheumatoid arthritis and otherautoimmune diseases mediated by T cells. In the embodiments of thepresent invention, compositions comprising said non-T cell bindingpeptides or their analogs and pharmaceutically accepted carriers oradjuvants are also provided.

The pharmaceutically accepted carriers or adjuvants include lactose,sucrose, sorbic alcohol, mannitol, potato starch, maize starch oramylopectin, cellulose derivatives, glutin, magnesium stearate, calciumstearate and so on. The compositions can be made in the form of tablet,pill, capsule, syrup, powder, granula or liquor and so on. For example,for oral administration of said pharmaceutical composition, the activecompound can be mixed with said pharmaceutically accepted carriers oradjuvants, and then impacted into tablets which can be further coated ifnecessary.

Liquor may also include other excipients well known in the art. Thepharmaceutical compositions can also include some pharmaceuticallyaccepted carriers, such as water, suspension agent, emulsifier and otheringredients.

The content of the non-T cell binding polypeptides or their analogs inthe compositions of the present invention are usually 50-200 μg/tabletand optimally, 100 μg/tablet. A person skilled in the art can determinethe particular amount of non-T cell binding polypeptides of the presentinvention to be administered according to the state of illness, bodyweight and age of patients.

The non-T cell binding peptides or their analogs of the presentinvention can be administered to arthritis patients by a variety ofmethods (oral administration, injection and so on).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The similarity of tridimensional conformations of five publishedHLA-DRβ1 binding polypeptides (CII, HSP70, Clip, HA and HB). The fiveoverlapped polypeptides are shown in different colors. The conformationsof these peptides are shown from different angles in the left and rightfigure. From the figure, it can be seen that the conformations of thesepolypeptides are extremely similar. The left side chains of amino acidsat position 1 (P1), position 4 (P4) and position 9 (P9) bind to HLA-DRβ1and are imbedded into the antigen binding clefts entirely or partially.The side chains of other amino acids stretch to another side or theorientation of TCR which is opposite to HLA-DRβ1 in order to stimulate Tcell activation. (Immunity 7:473-81,1997)

FIG. 2: The crystal structure of HLA-DRβ1 binding to CII polypeptide.The side chains at P1, P4 and P9 of CII polypeptide are imbedded intothe antigen binding “pocket”(s) of HLA-DRβ1. The polar binding pointwith high affinity is formed by (positively charged) Lys₇1 in HLA-DRβ1and P4 (Glu, negatively charged) (Immunity 7:473-81,1997).

FIG. 3: The comparison of the effects of non-T cell binding peptides andwild type CII polypeptide on T cell activation. Compared with wild typeCII peptide, none of polypeptides Mut 267-270, Mut 268-270, Mut 269-270,269A and 270A has significant effect on T cell activation.

FIG. 4: The inhibition effects of non-T cell binding peptides on T cellactivation. While co-incubating non-T cell binding peptides and wildtype peptides with T cell activation stimulation systems, the T cellactivation effects of wild type DRβ1 binding peptides can be inhibitedsignificantly by non-T cell binding peptides. The higher theconcentration of non-T cell binding peptides is, the heavier theinhibition effect is.

FIG. 5: The synovium pathological characters of CIA arthritis models.Incrassation synovium and lymphocye infiltration are found using HEstaining. Pannus formation is found in some joints. It is positivelycorrelated between the pathological changes and the ratio of jointswelling.

FIG. 6. The therapeutic effects of non-T cell binding peptide (267A) onCollagen-Induced Arthritis (CIA). On Day 8, 10, 12 and 14, the swellingratio(s) of right feet and arthritis score(s) of the three treatmentgroups are all significantly lower than those of the control group(p<0.05 or 0.01). On Day 8 and Day 16 after the treatment, the remissionratio(s) of CIA in the control group are significantly lower than thatin the three treatment groups (p<0.05).

FIG. 7. The Inhibition effects of non-T cell binding peptide (267A) oncollagen-induced arthritis (CIA) TNFα. The concentration of TNFα inperipheral blood in 100 μg/ml treated group is significantly lower thanthat of the control group (p<0.05). No significant variety is foundwhile comparing the other two treatment groups with the control group.

The following examples will be used to further illustrate the presentinvention, rather than limiting the scope of the present invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS EXAMPLE 1 PolypeptideDesign and Synthesis

It proves in the research mentioned above^((4,5,9-11)) that amino acidsP267-270 in CII polypeptide mainly bind to T cell receptors and activateT cells. In the experiments of the present invention, firstly, a groupof seven non-T cell binding peptides containing a single or multipleamino acid substitutions (CII 267-270) (table 2) were synthesized usingsolid-phase technique reported by Flechsler et al.⁽¹⁴⁾. In order toincrease the absorption ratio and the bioavailability of thesepolypeptides and enhance the therapeutic effect, the amino-terminal ofeach peptide was linked to a myristic acid gene in the process ofsynthesis to facilitate transportation of the polypeptides into cells.The ten-amino-acid-peptides used in this research all contain four ormore hydrophilic residues, such as Lysine (K) and Glutamate (E), and areeasy to be dissolved and absorbed. These peptides do not containmethionine (M) and tryptophan (W), which can be degraded easily, norcontains Aspartate (N) from which the amino group or the carboxyl groupis prone to be removed. Therefore, the peptides are stable, and it iseasy for them to enter into the cells and play a role in disturbing Tcell recognition. TABLE 2 Design of non-T cell binding peptides

It has been indicated by research that with substitution of T cellbinding amino acids (267-270) with Alanine (A) and/or Glycine (G)respectively, the aforementioned seven non-T cell binding peptides areable to suppress T cell activation. Among them, the significant effectsof polypeptide 267A and Mut 267-270 have been demonstrated in cell linetests and rheumatoid arthritis animal models (described later).

EXAMPLE 2 The Inhibition Effects of Non-T Cell Binding Peptides of thePresent Invention on T Cell Activation

Antigen polypeptides are expressed through antigen presenting cells andactivate T cells, resulting in the production of inflammation factors invivo, and further resulting in the typical pathologic changes ofrheumatoid arthritis. In the experiment, the inhibition effects of non-Tcell binding peptides on T cell activation were detected by measuring Tcell proliferation and Interleukin (IL)-2 level. Two sets of systems ofantigen presenting and T cell activation, which are recognizedinternationally, are used in the research. Antigen L57.23 cell:recognizing HLA-DR1 antigen presenting (transgened with DR1) cells:Priess cell: recognizing HLA-DR1/4 (transfected with EBV) T-cells: 3.19cell: HLA-DR1-specific T cell clone 3838 cell: HLA-DR4-specific T cellclone

Wherein one set of antigen presenting and T cell activation system iscomposed of L57.23 cell and 3.19 cell, and the other set of system iscomposed of Priess cell and 3838 cell. By comparing the difference inthe effects of said seven non-T cell binding peptides on antigenpresenting and T cell activation, non-T cell binding peptides which havesignificant inhibition effect(s) on T cell activation were screened.

Firstly, the antigen presenting cells, different non-T cell bindingpeptides (10 μg/ml) and corresponding T cells were added into thereaction system and co-incubated at 37° C. for 48 hours. The supernatantwas obtained and added into well-grown IL-2 dependent cells (CTLL). TheCTLL cell proliferation was detected by using titrazolium salt method(MTT). Thus the inhibition effects of non-T cell binding peptides on Tcell activation were determined.

The results of this research showed that wild type DRβ1 binding peptidewas able to stimulate T cell proliferation at a concentration of 400μg/ml, 200 μg/ml, 80 μg/ml, 40 μg/ml and 20 μg/ml, and highconcentration of IL-2 could be detected in the supernatant. However, thestimulation effects of the seven non-T cell binding peptides on T cellactivation were significantly weaker and the concentration of IL-2 inthe supernatant was significantly lower than that of wild type peptide(p<0.01) (FIG. 3). Non-T cell binding peptides can significantly inhibitthe T cell activation effects of wild type DRβ1 binding peptides whenthe non-T cell binding peptides were co-incubated with wild typepeptides and T cell activation system. Moreover, the effect ofinhibition increased with the increase of concentration of non-T cellbinding peptides (FIG. 4). Further tests proved that using non-T cellbinding polypeptides with substitution on amino acid 267 to treatcollagen-induced arthritis rats, the joint swelling and inflammatoryreactions could be significantly alleviated. The above results show thatthe wild type CII263-272 polypeptide contains the antigen epitope ofCollagen type II, and non-T cell binding peptides produced bysubstituting T cell binding amino acids of CII263-272 can block therecognition of T cell receptors to that polypeptide and thus inhibit Tcell activation. All these results suggest that non-T cell bindingpeptides might lead to a new method to interfere the autoimmuneresponses mediated by HLA-DR1/DR4.

EXAMPLE 3 The Inhibition Effects of Non-T Cell Binding Peptides of thePresent Invention on Experimental Arthritis

During the immune responses that lead to arthritis, antigen presentingcells induce T cell activation through antigen(s) and promote T cellproliferation, increase the in vivo cytokine (e.g. TNFα, IL-1) level inanimals and thereby lead to typical pathological changes of rheumatoidarthritis. Therefore, the inhibition effects of non-T cell bindingpeptides on arthritis can be determined by detecting the level of invivo cytokines in the experimental animals. The therapeutic effects canalso be evaluated through a pathological section (slice) from ahistological perspective.

In the animal experiment protocol of the present invention,collagen-induced arthritis (CIA) in Wistar rats was induced by bovineCII. The non-T cell binding peptides of the present invention were usedto treat the arthritis rats. It was found that polypeptide 267A couldsignificantly suppress arthritis in rats and alleviate the swelling ofjoints, and shorten the disease course (see the results).

(1) The Establishment of Experimental Arthritis Animal Model(s)

The experimental arthritis model used in the present invention iscollagen-induced arthritis (CIA) which is recognized internationally.The experimental animals used were American Wistar rats, which belong toa blocking colony. They were cultured successfully by Wistar Instituteof USA in 1907. They are among the species that were introduced into ourcountry earliest and were used most widely. The rats used in thisexperiment were all cultured in the animal center of People's Hospital,Peking University (SPF degree II). The rats were cultured in thepolyacylene cages cushioned by serrago. The lightness/darkness cycle wasmaintained as 12 h/12 h. The water and food were freely available to therats. All the rats used in the experiment were male, 8-10 weeks of age,body weight 180±10 g. All the protocols related to animals in theexperiment were in accordance with Chinese Experimental AnimalRegulations.

The bovine type II collagen (catalogue number: C11188) was bought fromSigma Co. Ltd.. It was dissolved in acetic acid to make a solution withthe final concentration of 8 mg/ml. Then the solution was emulsifiedwith complete Freund's adjuvant (Detroit, USA) of the equal volume. 100μl of the emulsified solution was injected into the footpad of the rightlimb of each Wistar rat. The injection dose was 400 μl/rat. Thus thearthritis model of Wistar rats was successfully established.

(2) The Evaluation of Arthritis

The swelling of metatarsophalangeal joints and interphalangeal jointswas found on all the rats about 15 days after the induction of arthritisin Wistar rats with CII. A 0-16 scale was used to evaluate the extent ofarthritis. Each of the 4 paws was assigned a score using a 0-4 scale,wherein zero=no joint swelling, 1=swelling of one joint, 2=swelling of 2joints, 3=swelling of 3 joints, and 4=swelling of 4 joints, which is theswelling of the whole paw. Four paws were scored respectively, and thesescores were summed up to give a total score. Beginning 2 days after theimmunization, the rats were subjected to an inspection every day untilthe experiment had been finished.

(3) Treatment of Arthritis in Wistar Rats by Non-T Cell BindingPeptide(s)

The arthritis rats were randomly divided into 4 groups from Day 4 to Day7 after arthritis induced therein. There were 3 treatment groups and onecontrol group. Each group consisted of 10 rats. Non-T cell bindingpeptide 267A was subcutaneously injected into the base of tail of eachrat every 3 days. The peptides were dissolved in sterile distilled waterbefore injection. The concentrations used in the 3 treatment groups were100 μg/ml, 500 μg/ml and 1000 μg/ml respectively, 100 μl for each rat,and the control group received sterile distilled water only. The volumechange of right feet was measured by water-expelling method every day.Using the aforementioned method of arthritis evaluation, the arthritisscores were given every day based on the number of joints witharthritis. The rats were killed 18 days after treatment. The peripheralblood was collected. The serum concentrations of the respectivecytokines were detected using ELISA kits of TNFα and IL-2 (JingMeiBiotech Co. Ltd, ShenZhen, P.R.C). In the meanwhile, the joints of theright limb were detached, fixed, decalcified, sectioned and subjected toHE staining.

(4) Statistical Analysis was Carried Out Using SPSS Software Package.

(5) Results: the Inhibition Effects of the Non-T Cell Binding Peptidesof the Present Invention on Experimental Arthritis.

40 male Wistar rats were subjected to induction of arthritis by bovinecollagen type II. As a result, metatarsophalangeal and interphalangealjoint swelling in different degrees appeared in all the rats from Day 14to Day 17. From Day 4 to Day 7 after the appearance of arthritis, therats were randomly divided into 4 groups, one of which was the controlgroup, while the other three were treatment groups. There were 10 ratsin each group. The polypeptide 267A concentrations used in the 3 groupswere 100 μg/ml, 500 μg/ml and 1000 μg/ml respectively. The scores weregiven based on the numbers of joints with arthritis by using theaforementioned method of arthritis evaluation.

As shown in the results, before the treatment, no significant differenceof arthritis scores on right foot swelling ratio and concentration ofIL-2 in peripheral blood was observed among the groups. HE staining ofall the swollen ankles and metatarsophalangeal joints showed synovialmembrane hyperplasia and lymphocyte infiltration in different degrees,and pannus formation was found in some joints (FIG. 5). The pathologicalchanges correlated positively with joint swelling. The swelling of theright feet of rats in the 3 treatment groups was alleviated within thefirst 2 weeks after treatment, while no significant change on theswelling of the right feet was found in the control group.

After the treatment of non-T cell binding peptide 267A of the presentinvention to the rats with arthritis, the arthritis of all the rats inthe 3 treatment groups began to be significantly alleviated on Day 4after the drug administration (FIG. 6, Table 3 as below). The remissionratios of the 3 treatment groups were significantly higher than those ofthe control group (P<0.05). On Day 8, 10, 12 and 14, both the right footswelling ratios and the arthritis scores of the 3 treatment groups weresignificantly lower than those of the control group (P<0.01); nosignificant difference among the 3 treatment groups was observed. TheTNFα concentrations in the peripheral blood of 100 μg/ml treated groupwere significantly lower than those of the control group (P<0.05, FIG.7). All the results prove that non-T cell binding peptides can reducethe auto-immune inflammation in the rats with arthritis, inhibit thecollagen-induced arthritis, and the peptides might have therapeuticeffects on rheumatoid arthritis. TABLE 3 The changes of right hind footswelling in rats of different groups Hind number foot of volume swollenNumber Ankle Toe1 Toe2 Toe3 Toe4 Toe5 (ml) joints Average control1 + + + + + + 1.5 6 6.2 group 2 − + ++ ++ ++ − 1.4 7 3 + ++ ++ + ++ −1.5 8 5 + − ++ + + + 1.3 6 6 + − + + + ++ 1.4 6 7 + − + ++ + + 1.3 6 8 +++ − + + + 1.5 6 9 + + + + + + 1.4 7 10 + + + − + − 1.2 4 10 mg 1 + −− + − − 1.2 2 3.15* group 2 − + + − + + 1.5 4 3 + + − − − + 1.4 3 4 − −− − + ++ 1.3 3 5 + − + − − ++ 1.3 4 6 + − + − − − 1.3 2 7 − + − − − −1.1 1 8 + + ++ − + + 1.3 6 9 + − − − − − 1.3 1 10 + ++ + − − − 1.2 4 50mg 1 + + + − + − 1.4 4 3.25* group 2 + + − ++ + + 1.4 6 3 + + + − + −1.4 4 4 − + − + ++ ++ 1.7 6 5 + − − − + − 1.3 2 6 + − − − − − 1.4 1 7 +− − − − − 1.1 1 8 − − + − − − 1.6 1 9 + + − − − + 1.6 3 10 − − − + + +1.4 3*The number of swollen joints significantly reduced compared with thecontrol group (p < 0.01).

The applicant also performed the aforementioned experimental research ofT cell activation assay and/or CIA models with other non-T cell bindingpeptides of the present application, including 268A, 269A, 270A, Mut267-270 and Mut 267-270, and the results achieved were similar to theresults of 267A.

REFERENCES

-   1 Gregersen P. K, et al.: Arthritis Rheum 30: 1250-1213, 1987.-   2 Wucherpfennig K W et al.: J Exp Med. 181:1597-1600, 1995.-   3 Zhanguo Li et al.: Chinese Journal of Immunology, 2002, 18: 76-78.-   4.Li Z G et al.: J Clin Invest (submitted) 2001.-   5 Li Z G et at.: Chin Med J 2002.-   6 Zhanguo Li et al.: Clinical Chinese Journal of Internal Medicine    2001, 40: 19-21.-   7 Zhanguo Li et al.: National Medical Journal of China 2001, 81:    111-113.-   8 Zhanguo Li et al.: Clinical Chinese Journal of Rheumatology 2001,    5: 145-147.-   9 Stern L J, et al.: Nature 368: 215-221, 1994.-   10 Fremont D H et al.: Science 272: 1001-1004, 1996.-   11 Jardetzky T S, et al.: Proc. Natl. Acad. Sci. USA93:734-738,    1996.-   12 Dessen A, et al.: Immunity 7:473-81,1997.-   13 Rosloniec E F, et al: J Exp Med.185: 1113-1122, 1997.-   14 Anderson E C, et al: Proc. Natl. Acad. USA 95: 7574-79, 1998.-   15 Flechsler I et al.: J Pept Scil (3): 141-200, 1995.

1. A non-T cell binding peptide and analogues thereof which bind to thetarget sequences having the consensus sequence of QK/RRAA (i.e.Gln-Lys/Arg-Arg-Ala-Ala)
 2. A non-T cell binding peptide which isselected from the group consisting of the following sequences:FKGEAGPKGE (SEQ ID NO: 1) FKGEQAPKGE (SEQ ID NO: 2) FKGEQGAKGE (SEQ IDNO: 3) FKGEQGPAGE (SEQ ID NO: 4) FKGEQGAAGE (SEQ ID NO: 5) FKGEQAGAGE(SEQ ID NO: 6) FKGEGAGAGE. (SEQ ID NO: 7)


3. A pharmaceutical composition comprising the non-T cell bindingpeptide(s) and analogues thereof according to claim 1, andpharmaceutically accepted carriers and adjuvants.
 4. The use of thenon-T cell binding peptide(s) and analogues thereof according to claim 1in the preparation of a medicament for the treatment of rheumatoidarthritis.
 5. The use of the non-T cell binding peptide(s) and analoguesthereof according to claim 1 in the treatment of rheumatoid arthritis.6. Administering the non-T cell binding peptide(s) and analogues thereofaccording to claim 1 to the patients with rheumatoid arthritis.
 7. Apharmaceutical composition comprising the non-T cell binding peptide(s)according to claim 2, and pharmaceutically accepted carriers andadjuvants.
 8. The use of the non-T cell binding peptide(s) according toclaim 2 in the preparation of a medicament for the treatment ofrheumatoid arthritis.
 9. The use of the non-T cell binding peptide(s)according to claim 2 in the treatment of rheumatoid arthritis. 10.Administering the non-T cell binding peptide(s) according to claim 2 tothe patients with rheumatoid arthritis.
 11. A peptide, which is obtainedby substituting one or more amino acids in position No.5-No.8 in theamino acid sequence of FKGEQGPKGE with Alanine and/or Glycine.
 12. Thepeptide according to claim 11, wherein the amino acid sequence of thesaid peptide is selected from the group consisting of the followingsequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7.
 13. A pharmaceutical compositioncomprising: (a) the peptide according to claim 11; and (b) apharmaceutically accepted carrier and/or adjuvant and/or excipient. 14.The pharmaceutical composition according to claim 33, wherein the aminoacid sequence of the said peptide is selected from the group consistingof the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ BD NO:7.
 15. The use of thepeptide according to claim 11 in the treatment of rheumatoid arthritis.16. The use according to claim 15, wherein the amino acid sequence ofthe said peptide is selected from the group consisting of the followingsequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7.